There are many use environments, the fields of medical research and pharmaceutical development being examples, where it is necessary to accurately acquire fluid samples with volumes that may be as small as a few nanoliters. In these same fields, it is also often desirable to measure optical characteristics of the acquired fluid samples. Such optical characteristics include, for example, the ability of a sample to absorb light.
For instance, UV-Visible Spectrophotometry may be used to characterize the chemical composition of a liquid sample (in solution or suspension phase) using the absorbed spectra of the sample. The light absorbance of a sample depends on the pathlength L of light passing through the sample, as well as on the concentration of light absorbers (e.g., biomolecules, cells, etc) in a sample solution and the wavelength (λ) of light being used to characterize the sample. The wavelengths of UV-Visible light span from 200 nm to 800 nm, while ultraviolet wavelengths range from 200 to 400 nm.
UV-Visible spectrophotometry provides a convenient analysis technique to determine the concentration, purity, and integrity of a biological sample without requiring additional sample preparation other than acquiring a sample. UV-Visible Spectrophotometry measurements depend on the light source (UV lamp), the sample and sampling technique. Most biological samples absorb electromagnetic radiation at wavelengths ranging from 200 nm to 800 nm, mostly 230, 260 and 280 nm. For a DNA or RNA sample in aqueous phase, one unit of absorbance 1 Å measured at a λ 260 nm and a pathlength of 10 mm is equal to 50/(40) ng/μl concentration.
Most biological samples are highly concentrated for downstream processing (such as microarray spotting or protein sample preparation for mass spectrometers). The absorbance of such samples can be above the saturation limit for typical spectrophotometers if the pathlength is about 10 mm. While the sample concentration range can be extended by diluting the sample, diluting sample requires additional laboratory work and can result in errors. Other approaches are needed to extend the sample concentration range that can be evaluated by the instrument.
Sampling techniques used in conventional UV-Visible Spectrophotometers include utilizing a cuvette with an optical window and fixed optical pathlength that holds a sample in a semi-closed way, direct measurement of liquid sample in a sample container (e.g., a well) along with a real-time pathlength measurement, and using a cuvetteless sample held in semi-free space between optical fibers which define a light path from a light source to a detector.
The cuvette-based sampling technique is widely used in conventional UV-Visible spectrophotometers. Generally, a sample is pipetted into a cuvette that has either a 10 mm or 2 mm path length. This technique is very limited for most biological samples since cuvettes typically used generally require a minimum 10 μl sample, which is problematic for valuable biological samples which may be present in limiting quantities, such as samples of protein or nucleic acids. A cuvette made of quartz or silica is expensive so it is typically reused after cleaning and drying. Further, adding 10 μl of sample with a pipette into a cuvette sometimes produces an air-bubble interface in the light path that can cause measurement error or void measurements. Additionally, a pathlength of 2 mm or 10 mm limits the sample concentration that may be measured to 1000 ng/μl for DNA/RNA sample due to the limited dynamic range of absorbance of most spectrophotometers.
Direct UV-Visible spectrophotometry measurement of liquid samples also suffers from limitations, such as the need to determine pathlength and adjust sample concentration. Pathlength depends on the sample well dimensions and sample volume. The determination of pathlength requires use of instruments such as level detectors or position sensors. For a pathlength ranging from 2 mm to 10 mm or above, the workable range of sample concentration for a spectrophotometer measurement becomes limited. For an example, for a DNA sample, if the pathlength is 10 mm, one unit of absorbance is equal to 50 ng/ul concentration (OD), and the upper limit of detection is typically 250 ng/μl if the upper limit absorbance of the spectrophotometer is 5. In this case, sample dilution is required for a sample concentration greater than 250 ng/μl. Sample dilution for multiple well plate measurements can be a complex laboratory operation.
Cuvetteless sampling also suffers from drawbacks. For example, in cuvetteless sampling, typically a narrow beam of light is directed to a sample stage that consists of a 1-2 μl liquid droplet suspended between two multi-mode optical fibers, one source-side fiber which provides light from a light source to the droplet and a detection-side fiber that guides light from the droplet to appropriate detection optics. The close proximity between the source-side and detection-side fibers allows enough of the light cone emanating from the source-side fiber to be collected by the detection-side fiber after passing through a liquid sample.
Cuvetteless instruments typically require a clamping surface that can be wetted with sample to avoid an air-bubble interface. Carry-over contamination is not completely removed with a simple wiping-off of the clamping surface. Adding a small amount of sample (1 μl) to the center of the clamping surface is also a complicated lab technique.
In summary, existing sampling techniques used in the conventional UV-Visible Spectrophotometers generally require too much sample, provide insufficient confidence in the sample application technique, may result in carry-over contamination, and may require pathlength determination and/or dilution of sample, over a range of solution concentrations.